State-of-the-art and preparatory work

There are two principal modes of translation initiation in eukaryotes – standard cap‐dependent (5’ end dependent) initiation as proposed by Kozak and so‐called internal initiation. In the latter case, 40S ribosomes recognize and bind to specific internal structures within 5’UTRs of mRNAs, termed IRES‐elements (Internal Ribosome Entry Sites), rather than to their 5’ends. The existence of this mechanism has been proven for genomic RNAs from viruses with positive‐strand RNA genomes (mostly for RNAs of picorna‐ and flaviviruses) (1). The mechanism of internal initiation has been deciphered to a significant extent only for IRES‐elements of some representatives of type II picornaviruses [encephalomyocarditis virus (EMCV), cardioviruses], foot‐and‐mouth disease virus (FMDV, aphthoviruses)) and for some flaviviruses (2‐6). This has been mostly done by the reconstitution of translation initiation complexes from purified components (2). The formation of translation initiation complexes on EMCV and FMDV IRESs has been found to require some additional mRNA‐binding proteins along with canonical initiation factors (see 1). For type I picornaviruses which include enteroviruses and rhinoviruses, respectively, the reconstitution of translation initiation complexes has not been achieved. Although specialists in the field are confident that these IRESs also require specific trans‐acting factors, their identity is still a matter of debate.

References

1. Pestova, T. V., Kolupaeva, V. G., Lomakin, I. B., Pilipenko, E. V., Shatsky, I. N., Agol, V .I. & Hellen, C.U.T. (2001) Molecular mechanisms of translation initiation in eukaryotes. Proc Natl Acad Sci USA 98, 7029‐7036.

2. Pestova, T.V., Hellen, C.U. & Shatsky, I.N. (1996). Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol 16, 6859‐6869.

3. Pestova, T.V., Shatsky, I.N., Fletcher, S.P., Jackson, R.J. & Hellen, C.U.T. (1998) A prokaryotic‐like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev 12, 67‐83.

4. Dmitriev, S.E., Terenin, Y.M., Dunaevsky, Y.E., Merrick, W.C. & Shatsky, I.N. (2003) Assembly of 48S translation initiation complexes from purified components with mRNAs that have some base pairing within their 5' untranslated regions. Mol Cell Biol 23, 8925‐8933.

5. Pisarev, A.V., Chard, L.S., Kaku, Y., Johns, H.L., Shatsky, I.N. & Belsham, G.J. (2004) Functional and structural similarities between the internal ribosome entry sites of hepatitis C virus and porcine teschovirus, a picornavirus. J Virol 78, 4487‐ 4497.

6. Terenin, I.M., Dmitriev, S.E., Andreev, D.E. & Shatsky, I.N. (2008) Eukaryotic translation initiation machinery can operate in a bacterial‐like mode without eIF2. Na. Struct Mol Biol 15, 836‐841.

7. Bung, C., Bochkaeva, Z., Terenin, I., Zinovkin, R., Shatsky, I. N. & Niepmann M. (2010) Influence of the hepatitis C virus 3'‐ untranslated region on IRES‐dependent and cap‐dependent translation initiation. FEBS Letters 584, 837‐842.

Aims

This project aims

I. to identify auxiliary trans‐acting factors which are necessary for formation of 48S translation initiation complexes on the IRES elements from RNAs of type I picornaviruses (polio‐ and rhinoviruses).

II. to extend the approach elaborated for picornaviuses to RNAs from other classes of animal viruses.

Work programme and methods

Specifically, this project aims to identify auxiliary trans‐acting factors which are necessary for formation of 48S translation initiation complexes on the IRES elements from RNAs of type I picornaviruses (polio‐ and rhinoviruses). To this end, a new method of identification of trans‐acting mRNA‐binding proteins recently developed in our lab will be applied. Once these auxiliary factors are identified, they will be included into the system of reconstitution of 48S translation initiation complexes on IRES‐elements from polio‐ or rhinovirus RNAs. A successful formation of 48S initiation complexes with these IRES elements will indicate that auxiliary factors are correctly identified. Moreover, this will open a way for dissecting in detail the mechanism of internal initiation used by these viral RNAs. In a broader sense, realization of this project will allow us to acquire a general approach for identification of proteins that modulate the activity of 5’UTRs of eukaryotic mRNAs.

State of research

The method of reconstitution of 48S translation initiation complexes from totally purified components has been proposed and developed in this lab (2, 4). Using this approach, the proteins bound to IRES‐elements from several representatives of picorna‐ and flaviviruses have been identified (2‐6). However, attempts to extend these approaches to poliovirus IRES were unsuccessful. The main reason was a low efficiency of formation of translation initiation complexes on these IRESs even in cellular extracts. This precluded accumulation of sufficient material to identify key auxiliary factors that determine the translation initiation on polio‐ and rhinovirus IRES‐elements. Addition of some individual mRNA‐binding proteins reported to be involved in the translation initiation with these IRES was unsuccessful either, especially as the identity of these key auxiliary proteins is still controversial.

Previous and planned work

We have recently found two requirements to obtain a rather high yield of 48S complexes on the poliovirus IRES‐element: 1) the reconstitution of 48S complexes should be performed in the rabbit reticulocyte system with addition of extract from HeLa cells; 2) a model RNA containing the poliovirus IRES should have a poly(A) tail at the 3’end. The basic protocol to isolate specific proteins bound to IRESs is as follows: the RNA to be employed in the formation of 48S complexes is slightly biotinylated at U residues in the course of its synthesis in the T7 transcription system. After separation of 48S complexes from unbound RNA by sucrose gradient sedimentation, they are mixed with streptavidin agarose adsorbent. The adsorbent is then washed with appropriate buffer and proteins bound to mRNAs are eluted from agarose by treatment with a ribonuclease. The eluted proteins are separated by gel electrophoresis, cut out from the gel and identified by massspectrometry. The use of Ca‐dependent micrococcal nuclease instead of ribonuclease A will allow us to use the eluted proteins directly for the reconstitution of 48S complexes from individual components.

Titles for dissertations (prospective)

• Molecular mechanisms of translation initiation on poliovirus and rhinovirus IRES‐elements

• Functional role of cellular trans‐acting factors in the translation initiation on picornavirus IRESelements

Relationships/connections within the research training group

Niepmann: Trans‐acting factors in the translation initiation on viral RNAs

Bindereif: Exchange of methodological expertise in studying RNA‐protein interactions by different approaches in vivo and in vitro

Ziebuhr: Analysis of the molecular mechanism of translation initiation on RNAs from coronaviruses.

Benefits of the scientific exchange

The group plans to continue its collaboration with the Niepmann lab. This collaboration turned out to be very fruitful and resulted in a recent common publication. The benefit of scientific cooperation is accounted for by quite similar scientific interests and the necessity to prepare a set of protein factors needed for reconstitution of translation initiation complexes, the principal technique used in the studies. Isolation of protein factors is a costly and time‐consuming procedure. It strongly requires combined efforts of two or more labs. The future cooperation with the Ziebuhr group will extend the expertise of Shatsky group in studies of viral mechanisms of translation initiation to RNAs from a quite different class of viruses, coronaviruses. Some methodological cooperation with the Bindereif group on studies of RNA‐protein interactions in splicing is also planned.